Analysis of signal transduction and protein phosphorylation is fundamental to understanding physiological and pathological cell behavior and identifying novel therapeutic targets. Despite the fact that the use of physiological three-dimensional cell culture assays is increasing, 3D proteomics and phosphoproteomics remain challenging due to difficulties with easy, robust and reproducible sample preparation. Here, we present an easy-to-perform, reliable and time-efficient method for the production of 3D cell lysates that does not compromise cell adhesion before cell lysis. The samples can be used for western blotting as well as phosphoproteome array technology. This technique will be of interest for researchers working in all fields of biology and drug development.